Pectic substance as a growth factor stabilizer

ABSTRACT

Pectic substance from Aloe Vera and other sources is used as a stabilizer and a delivery vehicle for pectin/heparin-binding proteins, such as pectin/heparin binding growth factors. Aloe pectin, a naturally occurring LM (low methoxyl) pectin, binds to pectin/heparin-binding growth factors, i.e., bFGF, aFGF, and KGF of fibroblast growth factor (FGF) family and TGF-β1 of transforming growth factor-β (TGF-β) family. Commercial LM or HM (high methoxyl) citrus pectins tested did not exhibit any binding activity with bFGF. A weak binding to bFGF was observed with a de-esterified pectin (polygalacturonic acid) prepared from citrus. The binding protected the growth factor from protease digestion. The calcium gel beads prepared with Aloe pectin also bound to these pectin/heparin-binding growth factors. The growth factor could also be encapsulated in the pectin calcium gel and Aloe pectin sodium gel. Pectin/heparin-binding growth factor stabilized by pectin is used for wound healing. A pectin-containing matrix is used for the isolation of a pectin/heparin-binding protein.

This is a continuation-in-part of U.S. patent application Ser. No.09/078,204, filed May 13, 1998 U.S. Pat. No. 5,929,051.

BACKGROUND

This invention relates to using a pectic substance to stabilize apectin/heparin-binding protein, the resultant composition andformulation, and the method of stabilizing the pectin/heparin-bindingprotein.

Following abbreviations are used in this invention:

aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growthfactor; BSA, bovine serum albumin; Da, dalton; DAc, degree ofacetylation; DM, degree of methylation; EDTA, ethylenediaminetetraaceticacid; EGF, epidermal growth factor; Gal A, galacturonic acid; HM, highmethoxyl; HMW, high-molecular-weight; IL, interleukin; kDa, kilodalton;KGF, keratinocyte growth factor; LM, low methoxyl; LM,low-molecular-weight; PDGF, platelet-derived growth factor; SDS, sodiumdodecyl sulfate; TGF-α, transforming growth factor-α; TGF-β1,transforming growth factor-β1; TN buffer, 25 mM Tris, 0.15M NaCl, pH7.4; TNF-α, tumor necrosis factor-α.

Pectin is a plant cell wall component. The cell wall is divided intothree layers, middle lamella, primary, and secondary cell wall. Themiddle lamella is the richest in pectin. Pectins are produced anddeposited during cell wall growth and are particularly abundant in softplant tissues under conditions of fast growth and high moisture content.In cell walls, pectins are present in the form of a calcium complex. Theinvolvement of calcium cross-linking is substantiated by the fact thatchelating agents facilitate the release of pectin from cell walls.

Pectin is a complex polysaccharide. It consists of an α1-4 linkedpolygalacturonic acid backbone intervened by rhamnose residues andmodified with neutral sugar side chains and non-sugar components such asacetyl, methyl, and ferulic acid groups. The neutral sugar side chainswhich include arabinan and arabinogalactans (Types I and II) areattached to the rhamnose residues in the backbone at the O-3 or O-4position. The rhamnose residues tend to cluster together on thebackbone. So with the side chains attached this region is referred asthe hairy region and the rest of the backbone is hence named the smoothregion. Rhamnose residues are 1-2 linked to Gal A residues in thebackbone and the configuration of this linkage has now been determinedto be α.

Because of the presence of neutral sugar side chains and some othernon-sugar components, the structure of pectins is very complex;essentially no two molecules have identical structures.

Rhamnose, galactose, arabinose, and xylose are the most common neutralsugar components of pectins. The less common ones are glucose, mannose,and fucose. Some of the xylose residues are individually attached to GalA residues at the O-3 position. Three types of neutral sugar side chainshave been identified in pectins. Arabinan consists of α1-5 linkedarabinose. Arabinogalactan I consists of β1-4 linked galactose withshort arabinan chains attached at O-3. In arabinogalactan II, galactoseis β1-3&6 linked with arabinose attached.

Methylation occurs at carboxyl groups of Gal A residues. The degree ofmethyl-esterification is defined as the percentage of carboxyl groups(Gal A residues) esterified with methanol. A pectin with a degree ofmethylation (“DM”) above 50% is considered a high methoxyl (“HM”) pectinand one with a DM<50% is referred to as low methoxyl (“LM”) pectin. Mostof the natural pectins are HM with a few exceptions such as sunflowerpectin. The degree of acetylation (DAc) is defined as the percentage ofGal A residues esterified with one acetyl group. It is assumed that onlythe hydroxyl groups are acetylated. Since each Gal A residue has morethan one hydroxyl group, the DAc can be above 100%. DAc is generally lowin native pectins except for some such as sugar beet pectin.

Generally, pectins are soluble in water and insoluble in most organicsolvents. Pectins with a very low level of methyl-esterification andpectic acids are only soluble as the potassium or sodium salts. As forother polymers, there is no saturation limit for pectins, but it isdifficult to obtain a true solution with concentrations higher than3-4%. Commercial pectins have a size range of 7-14×10⁴ Da with citruspectins being larger than apple pectins. Viscosities of pectin solutionsare generally low and so pectins are seldom used as thickening agents.The viscosity is directly related to the size, pH, and also to thepresence of counterions. Addition of monovalent cations reducesviscosity.

The Gal A residues in the pectin backbone are α1-4 linked. Both hydroxylgroups of D-Gal A at carbon atoms 1 and 4 are in the axial position. Theresulting linkage is therefore trans 1-4. This type of linkage resultsin increased chain stiffness of the polymer. So pectin with aflexibility parameter B between 0.072-0.017 are rigid molecules. It hasbeen suggested that the insertion of rhamnose residues in the backbonecause a T-shaped kink in the backbone chain. An increase in rhamnosecontent leads to more flexible molecules. Pectins can be considered as azigzag polymer with long and rigid smooth regions and flexible hairyregions (rich in rhamnose) serving as rotating joints. The DM also hascertain effects on chain flexibility. In solution, pectin molecules havebeen shown to assume a right-handed helical structure.

Pectins are most stable at pH 3-4. Below pH 3, methoxyl and acetylgroups and neutral sugar side chains are removed. At elevatedtemperatures, these reactions are accelerated and cleavage of glycosidicbonds in the galacturonan backbone occurs. Under neutral and alkalineconditions, methyl ester groups are saponified and the polygalacturonanbackbone breaks through β-elimination-cleavage of glycosidic bonds atthe non-reducing ends of methoxylated galacturonic acid residues. Thesereactions also proceed faster with increasing temperature. Pectic acidsand LM pectins are resistant to neutral and alkaline conditions sincethere are no or only limited numbers of methyl ester groups.

Both HM and LM pectins can form gels, but by totally differentmechanisms. HM pectins form gels in the presence of high concentrationsof co-solutes (sucrose) at low pH. LM pectins form gels in the presenceof calcium. In addition, the sugar beet pectin can form gels throughcross-linking of the ferulated groups.

The calcium-LM pectin gel network is built by formation of the “egg-box”junction zones in which Ca++ ions cause the cross-linking of twostretches of polygalacturonic acids. In apple and citrus pectins,stretches of polygalacturonic acids without rhamnose insertion have beenestimated to be as long as 72-100 residues. The zone is terminated bythe rhamnose residue in the backbone. The calcium-LM pectin gel isthermoreversible. The calcium can therefore be added at the boilingpoint and gel formation occurs upon cooling. It is possible to obtain afirm resilient gel with 0.5% pectin and 30-60 mg/g Ca++. A high contentof pectin with little calcium gives an elastic gel; whereas, a highcalcium concentration with a minimum of pectin results in a brittle gel.

Industrial pectins, either HM or LM, are mainly obtained from apple andcitrus by acid extraction and alcohol precipitation. LM pectins areobtained from HM ones by chemical de-esterification. Pectins have afavorable regulatory status as a food additive. They are classified asGenerally Recognized As Safe (“GRAS”) in the United States andAcceptable Daily Intake (“ADI”) in Europe. That is, its use is onlylimited by current Good Manufacturing Practice (“cGMP”) requirements tomeet certain specifications. These specifications include a minimal GalA content of 65% (w/w).

Many other plant sources have also been examined for pectin production.Two of them, sugar beet pulp and sunflower head, have been studiedextensively. Both are abundant as raw materials. However, sugar beetpectin has a poor gel forming ability largely due to its high acetylgroup content and small molecular size (˜5×10⁴ Da). The sunflowerpectins are naturally LM and can be efficiently extracted with chelatingagents. Chemically, sunflower head pectin has a very high Gal A contentand is a natural LM pectin, whereas sugar beet pectin has a relativelylow Gal A content and a very high content of acetyl and ferulic acidgroups. The structures of apple and citrus pectins are very similar toeach other. Pectins are traditionally used as food additives. However,their use has extended into pharmaceutical areas as well. They oftensuffer from poor quality of raw materials and poor color quality(usually tan) of the pectin end products.

Pectins from different plant sources have different characteristics. Ingeneral, all commercial pectins including those that have gone throughfurther processing have a certain degree of coloration as a finalproduct. The color ranges from light yellow/brown (citrus pectin) todark tan (apple and sunflower head pectins). The coloration is caused bythe combination of two factors: natural color (pigmentation) of the rawmaterials and their content of polyphenols.

Pectins are effective against gastrointestinal ulcers and enterocolitis.Pectins also influence cell proliferation in the intestines. They alsohave a blood cholesterol-lowering effect and exhibit inhibition ofantherosclerosis. This effect is the result of interactions betweenpectins and bile salts. Pectins have also been shown to affect thefibrin network in hypercholesterolaemic individuals. Pectins are alsoeffective in removing lead and mercury from the digestive tract andrepiratory organs.

Proteins possess unique chemical and physical properties which posedifficult stability problems: a variety of degradation pathways exist ofproteins, involving both chemical and physical instability. Thesemacromolecules are also at risk from microbial degradation due toadventitious contamination of the solutions during purification orstorage. All these considerations are especially critical for thepharmaceutical manufacturer who is formulating and packaging theseagents. Thus, a thorough pre-formulation program is an essential stepfor protein drugs, to solve their possible formulation problems.

SUMMARY

The present invention relates to composition and method of manufactureof a stabilized pectin/heparin-binding protein having apectin/heparin-binding protein and a pectic substance in an amounteffective to stabilize the pectin/heparin-binding protein. Usefulpectin/heparin-binding protein includes pectin/heparin-binding growthfactors. Useful pectic substance includes Aloe pectin, low methoxylpectin, de-esterified pectin, pectic acid, pectate, pectinic acid,pectinate, pectin calcium gel, Aloe pectin sodium gel, Aloe Vera innergel cell wall fiber, and others.

In one embodiment, the present invention provides a pharmaceuticalformulation which comprises a pectic substance, a pectin/heparin-bindinggrowth factor and a thickener.

In another embodiment, the present invention provides a method ofisolating a pectin/heparin-binding protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a-b. show the binding of Aloe pectin to bFGF. The binding assaywas carried out with pectin-rich Aloe Vera inner gel cell wall fibers asdescribed in Example 6. Soluble Aloe pectin (FIGS. 1a and 1 b) orheparin (FIG. 1b) was used as the inhibitor. A sample of bFGF (0.5 μg)was directly loaded onto the gel as a standard for comparison of the ODvalues (FIG. 1a), which was not a part of the binding or inhibitionreaction as indicated by “x.”

FIG. 2. shows a comparison of Aloe pectin with other pectins of variousDM values in inhibiting the binding of bFGF to the pectin-rich Aloe Verainner gel cell wall fibers.

FIG. 3. shows the protection of bFGF by Aloe pectin against enzymedigestion. bFGF (0.5 μg) was mixed with trypsin (0.5 μg) in the presenceor absence of Aloe pectin as described in Example 8. Followingincubation at 37° C. for 1 hr, bFGF was separated by SDS-polyacrylamidegel electrophoresis.

FIG. 4. shows the binding of Aloe pectin calcium gel beads to bFGF. Thebinding assay was carried out as described in Example 9. One gel beadwas used for each binding assay. Soluble Aloe pectin was used as aninhibitor.

FIG. 5. shows the affinity chromatography with Aloe Vera inner gel cellwall fiber as a pectin-containing matrix. bFGF was loaded onto a columnconsisting of inner gel cell wall fibers. Elution was performed stepwisewith 0.4 ml of NaCl at various molar concentrations. Fractions weresubjected to gel electrophoresis for detection of bFGF.

DETAILED DESCRIPTION

Thus, the general term “pectic substance,” as used in this invention,includes pectin, low methoxyl pectin, de-esterified pectin, pectincalcium gel, Aloe pectin sodium gel, pectic acid, pectate, pectinicacid, pectinate, protopectin, and pectin-rich substances, such as AloeVera inner gel cell wall fiber, individually, collectively, or incombination thereof. As discussed above, pectin is a group designationfor those complex colloidal carbohydrate derivatives which occur in, orare prepared from, plants and contain a large proportion ofanhydrogalacturonic acid units which are thought to exist in achain-like combination. The carboxyl groups may be partially esterifiedby methyl groups and partially or completely neutralized by one or morebases. Thus, “de-esterified” usually means that one or more methyl estergroups have been removed from the pectin molecules. “Pectic acids” isthe group designation applied to pectic substances mostly composed ofcolloidal polygalacturonic acids and essentially free from methyl estergroups. The totally de-esterified pectin is pectic acid orpolygalacturonic acid. “Pectates” are either normal or acid salts ofpectic acids. “Pectinic acids” are the colloidal polygalacturonic acidscontaining more than a negligible proportion of methyl ester groups.“Pectinates” are either normal or acid salts of pectinic acids.“Protopectin” is applied to the water-insoluble parent pectin whichoccurs in plants and which upon restricted hydrolysis yields pectins,pectinic acids, and others. The water-insoluble pectin may be associatedwith the cellulose present in the plant, such as the Aloe Vera inner gelcall wall fiber.

Aloe Pectin

Aloe Vera leaves consist of two parts, an outer green rind and a clearinner gel which is also referred as pulp. Aloe pectin is extracted fromthe inner gel or outer rind cell wall fibers. Use of a chelating agentat a slight alkaline pH is found to be the most efficient extractionmethod. Aloe pectin is unique as compared to previously describedpectins. It has a high rhamonse content, >4% in the purified pectinpreparation which is at least 2 times higher than in other pectins suchas citrus, apple, sugar beet, and sunflower. Rhamnose is a key sugar inthe pectin backbone whose content affects the flexibility of themolecule. Aloe pectin also possesses a rare sugar, 3-OMe-rhamnose whichhas not been described in any other pectins. Aloe pectin is naturallyLM, having a DM generally <30% and often <10%. The Gal A content of Aloepectin is >70%. Aloe pectin is capable of gel formation in the presenceof calcium. Uniquely, Aloe pectin, especially the high-molecular-weightHMW) one, can also form a monovalent cation-based reversible gel at lowtemperature (4° C.) at a pectin concentration as low as 1 mg/ml. Anexample of the monovalent cation is sodium. Such cold gelation has notbeen described for any other pectins.

Aloe pectin extracted from the inner gel fibers is an off white powderand produces a clear solution as compared to the yellow to tan powdersand cloudy solutions of current commercial and experimental pectins fromcitrus, apple, sugar beet, or sunflower.

Aloe pectin can be distinguished from other pectins by one or more ofthe following characteristics:

1) A high molecular weight (>1×10⁶ Da) and a high intrinsic viscosity(>550 ml/g).

2) A high rhamnose content (>4%).

3) Containing 3-OMe-rhamnose.

4) Being naturally LM.

5) Capable of calcium gel formation.

6) Capable of monovalent cation-based gel formation at low temperature(4° C.).

7) Off white powder and clear solution (Aloe pectin extracted from innergel fibers).

Pectin/Heparin-Binding Proteins

In the mammalian system, there are over 100 heparin-binding proteins.These include: (1) cellular matrix proteins, such as collagen,fibronectin, and vitronectin; (2) cytokines such as IL-3, IL-8, IFN-γ,PF-4, MIP-1, and members of fibroblast growth factor and transforminggrowth factor-β families; (3) enzymes such as elastase and phospholipaseA2; (4) proteins involved in blood clotting such as antithrombin,thrombin, and fibrin; and others.

There is a large number of cytokines that are essential regulators ofvarious biological functions in a mammal. They are usuallylow-molecular-weight proteins or peptides. Some of these cytokines aregrowth factors. Growth factors are proteins or peptides that are capableof stimulating cell division. They are important factors in cellproliferation and development. There are five major growth factorfamilies, epidermal growth factor (EGF), transforming growth factor-β(TGF-β), platelet-derived growth factor (PDGF), insulin-like growthfactor (IGF), and fibroblast growth factor (FGF). Growth factors alsoare important in wound healing. bFGF and TGF-β have been widely studiedfor accelerating wound healing. As used in this invention, the term“growth factor” denotes a natural and/or a recombinant growth factor,purified or unpurified, in vitro, in vivo, or ex vivo.

Many growth factors are heparin-binding, including members of the TGF-βand FGF families. This binding is crucial for their functions. Heparinis a polymer of a sulfated disaccharide repeat sequence(→GlcAβ1-4GlcNAcα1→) (Casu, (1989), Annals of New York Academy ofScience 556, pp. 1-17). It is derived from the polysaccharide chains ofproteoglycans commonly found on cell surfaces, i.e., glycosaminoglycan(GAG). Like pectin, heparin is a negatively charged molecule. Theinteraction of heparin with heparin-binding proteins, including acidicor basic fibroblast growth factor (aFGF or bFGF), has been extensivelystudied. The heparin-binding domains on aFGF and bFGF have beenidentified. Such domains always involve positively charged amino acidresidues, which play a major role in mediating the binding to theheparin through ionic interaction (Cardin and Weintraub, (1988),Arteriosclerosis 9, pp. 21-32; Faham et al., (1996), Science 271, pp.1116-1120). The minimal binding site on heparin for some of these growthfactors has also been identified. The one for bFGF has been shown toconsist of only five or six sugars. Due to variation in the extent andposition of sulfation, the protein binding specificities may bedifferent among different regions of a single heparin molecule. That is,different heparin-binding proteins may bind to different regions ofheparin molecules.

The binding to heparin stabilizes growth factors against inactivation byheat, acid, and protease digestion and provides a growth factorreservoir and a bioavailability control mechanism. The heparin-bindingalso facilitates the binding of growth factors to their high-affinitysignaling receptors and induction of the cross-linking of the receptorsfor initiating signal transduction.

Aloe Vera inner gel, a water storage tissue, consists of large mesophyllcells that contain only a very limited number of degenerated cellularorganelles. Following disruption of the inner gel by homogenization,mesophyll cell wall fibers (broken mesophyll cell wall fragments) can beisolated by low-speed centrifugation or filtration. These fibers appearto be clear transparent sheets under microscope. They are rich in Aloepectin (50%, w/w) which can be extracted or solubilized with thechelating agent EDTA.

The inner gel cell wall fibers could be readily pelleted by low-speedcentrifugation. Thus, binding assays for determining if Aloe pectinbinds to any growth factors, cytokines, or other proteins could beperformed using these fibers. The actual binding to Aloe pectin wasconfirmed by an inhibition assay using the soluble Aloe pectin. Theresults showed that Aloe pectin bound to heparin-binding growth factors,aFGF, bFGF, KGF of FGF family, and TGF-β1 of TGF-β family, but not toany non-heparin-binding proteins tested. The binding to heparin-bindingproteins, however, was selective since Aloe pectin did not bind toseveral other heparin-binding proteins tested. The binding was inhibitedby soluble Aloe pectin, thus confirming that the binding to thepectin-rich cell wall fibers was actually mediated by the pectinmolecules. The binding was also inhibited by heparin, being consistentwith the heparin-binding nature of these growth factors. The commercialLM or HM pectins tested did not exhibit any binding activity as shownwith bFGF. However, a weak binding to bFGF was observed with ade-esterified citrus pectin (i.e. polygalacturonic acid).

The binding protected the growth factor from protease digestion asdemonstrated with bFGF. Both soluble Aloe pectin and pectin-rich innergel cell wall fibers exhibited the protection effect. A protectioneffect, although weaker, was also observed with the polygalacturonicacid from citrus. This indicates that pectin can be used as a stabilizerfor these pectin/heparin-binding growth factors. As used in thisinvention, a heparin-binding proteins that also binds to a pectin istermed “pectin/heparin-binding protein.” Thus, a “pectin/heparin-bindinggrowth factor” is a growth factor that binds to a heparin and also bindsto a pectin. The calcium gel beads prepared with Aloe pectin also boundto these pectin/heparin-binding growth factors. Thus, the pre-formedpectin beads can be loaded with such growth factors for deliverypurposes. In addition, the growth factors could also be encapsulated inthe pectin calcium gel bead for delivery purpose. Furthermore, thesegrowth factors can also be entrapped in the monovalent cation-based gelas a storage form, which has an added advantage of preventing aggregateformation and permitting the eventual use in a solution for m. Thus,pectin can be used in various forms for stabilization and delivery ofpectin/heparin-binding growth factors, i.e., a solution, a pre-formedcalcium gel, a calcium gel formed in the presence of the growth factor,and a monovalent cation-based gel formed in the presence of the growthfactor. Lastly, the pectin-rich inner gel fiber can also be used as astabilizer for the pectin/heparin-binding growth factors.

The binding strength of these growth factors to Aloe pectin was assessedby affinity chromatography using inner gel cell wall fiber as apectin-containing matrix and bFGF as the binding ligand. The resultsshowed that the binding strength to Aloe pectin was moderate (0.5-0.7MNaCl) and weaker than that (1.4-1.6 M NaCl for bFGF) to heparin(0.3-0.4M NaCl is considered weak binding and >1.0M NaCl is consideredstrong binding; Conrade, (1998), Heparin-binding Proteins, AcademnicPress, San Diego, pp. 197-199). The weaker affinity to Aloe pectin mostlikely makes it even more suitable for using Aloe pectin as a stabilizerand delivery vehicle; once the growth factor in complex with pectinmakes contact with the glycosaminoglycan (heparin) on the cell surface,it will bind to the latter, its natural higher-affinity ligand, toinitiate a stimulatory effect on the cells.

TABLE 1 Interaction of Aloe pectin with heparin-binding growth factors.Proteins Binding to Aloe pectin Heparin-binding bFGF Yes aFGF Yes KGFYes TGF-β Yes Fibronectin No IL-8 No IFN-γ No Non-heparin-binding TGF-αNo PDGF No TNF-α No EGF No IL-1β No Complement C3 No BSA No

MATERIALS

Aloe Vera (Aloe Barberdensis Miller) plants (10″) were obtained from H&Psales, Inc (Vista, Calif.) through Lowe's store. Bulk acetylated mannan(“BAM” also known as acemannan hydrogel) is an Aloe Vera inner gelextract of Carrington Laboratories, Inc. Various commercial pectins andpolygalacturonic acid were used. They include HM citrus (P-9561 with aDM of 92% and P-9436 with a DM of 64%), LM citrus (P-9311 with a DM of28%), polygalacturonic acid (P-1879) from Sigma Chemical Co., HM citrus(PE100 with a DM of 67%) from Spectrum Chemical Co., and HM citrus(CU401) and apple (AU201) from Herbstreith-Fox KG. Following reagentswere also obtained from Sigma Chemical Co.; disodium EDTA, tetrasodiumEDTA, endo-polygalacturonase, and all neutral and acidic sugars used.The alkaline phosphatase substrate pNPP was obtained from Pierce. Sodiumhexametaphosphate was obtained from Fluka Chemie AG.

Recombinant growth factors/cytokines aFGF (acidic fibroblast growthfactor), bFGF (basic fibroblast growth factor), KGF (keratinocyte growthfactor), EGF (epidermal growth factor), TGF-α (transforming growthfactor-α), TNF-α (tumor necrosis factor-α), and IL-8 (interleukin-8)were obtained from Promega (Madison, Wis.). Recombinant TGF-β1(transforming growth factor-β1), IFN-γ (interferon-γ), PDGF(platelet-derived growth factor), and IL-1β (interleukin-1β) wereobtained from R&D systems (Minneapolis, Minn.). Fibronectin was obtainedfrom Gibco-BRL. Complement C3, bovine serum albumin (BSA), and heparinwere obtained from Sigma Chemical Co (St. Louis, Mo.). All the proteinswere of human origin except for TGF-β1 which is of porcine origin.

Generally, BAM may be prepared from Aloe leaves as follows:

1. Aloe leaves are washed, sliced open and filleted to remove the leafrind. The clean (substantially anthraquinones free) inner gel isretained while the green rind is discarded.

2. The filleted material is homogenized (creparo) and extensivelyfiltered with a Finisher Model 75 (FMC, Chicago, Ill.) to remove most ofthe fiber.

3. The clear viscous gel is acidified to a pH of approximately 3.2 withdilute HCl.

4. The acidified gel is then extracted with four volumes of 95% ethanolat ambient temperature. Floating material is removed, then thealcohol/water mixture is siphoned off while the solid precipitate iscollected by centrifugation. Most alcohol/water soluble substances suchas organic acids, oligosaccharides, monosaccharides, anthraquinones andinorganic salts are eliminated by the alcohol extraction process.

5. The solid Aloe Vera extract is then washed with fresh alcohol,centrifuged, freeze dried, and ground to a white powder.

The product is stable at room temperature in the freeze-dried form forseveral years if protected from additional moisture. The detailedprocedures for producing substantially anthraquinone-free Aloe gel, forproducing substantially anthraquinone-free Aloe juice, for extractingactive chemical substance(s) from an Aloe leaf, for preparing BAM andfor extracting from an Aloe leaf substantially non-degradablelyophilized ordered linear polymers of mannose have been described inCarrington's U.S. Pat. Nos. 4,735,935, 4,851,224, 4,917,890, 4,957,907,4,959,214, and 4,966,892, the entire content of each of which isincorporated by reference. The uses of Aloe products have been describedin Carrington's U.S. Pat. Nos. 5,106,616, 5,118,673, 5,308,838,5,409,703, 5,441,943, and 5,443,830, the entire content of each of whichis hereby incorporated by reference.

A dispersant (or a protective colloid, a suspending agent, a stabilizer,or an emulsifier) can be selected from a wide variety of materials.Non-limiting examples include glycerine, a polyvinylpyrrolidone (“PVP”),or a PVP K homopolymer, such as: PVP K-15 powder having an averagemolecular weight (“Mv”) of 8000 Daltons; PVP K-30 powder, Mv 38,000Daltons; PVP K-60, 45% solution, Mv 216,000 Daltons; PVP K-90 powder, Mv630,000 Daltons; or PVP K-120 powder, Mv 2,900,000 Daltons. Otherdispersants include a series of vinylpyrrolidone (“VP”)/vinyl acetate(“VA”) copolymers, which copolymers covering a range of VP/VA moleratios [given in bracket], suppled as either ethanol solution (“E”),isopropanol solution (“I”), or solid (“S”), PVP/VA E-735 [70/30], PVP/VAE-635 [60/40], PVP/VA E-535 [50/50], PVP/VA E-335 [30/70], thecorresponding isopropanol solution and PVP/VA I-235 [20/80], PVP/VAS-630 [60/40]. Other dispersants include: Polyvinylpyrrolidone,pharmaceutical grade, known as Povidone USP or Polyvidonum, some ofwhich are supplied as Plasdone C-15, Plasdone C-30, Plasdone K-25,Plasdone K-25, Plasdone K-26/28. Plasdone K-29/32. Plasdone K-90, orPlasdone K-120. Another class of dispersant is Crospovidone NFPolyvidonum insoluble, crosslinked N-vinyl-2-pyrrolidone. Still otherdispersant includes: Poly(methyl vinyl ether/maleic anhydride) (linearinterpolymer with 1:1 molar ratio) Series of copolymers, supplied asGantrez® AN-119, having molecular weight as determined by membraneosmometry (“M.Wt.”) of 20,000, Gantrez® AN-139 M.Wt. 41,000, Gantrez®AN-149 M.Wt. 50,000, and Gantrez® AN-169 M.Wt. 67,000.

The dispersant is sometimes termed a protective colloid, a suspendingagent, or an emulsifier.

Exemplary thickeners useful in this invention include: Those which areorganic in nature (such as karaya gum, acacia gum, tragacanth,methylcellulose, hydroxymethylcellulose, and sodiumcarboxymethylcellulose) or synthetic polymers (such as polyethyleneoxide, vinyl methyl ether/maleic anhydride compounds, cationicpolyacrylamide compounds, or acetic polyvinyl). Many of these arelargely carbohydrates or carbohydrate-like and swell with the additionof water. Preferably the thickeners are hydroxyethylcelluloses, such asNatrosol H (38000 cp), Natrosol 250 G Pharm (150-200 cp), and Natrosol250 GL (75-150 cp).

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. All literature citations are incorporated byreference.

EXAMPLE 1 Extraction of Aloe Pectins

Preparation of cell wall fibers

Two types of cell wall fibers were used, alcohol-treated andnon-alcohol-treated. The alcohol-treated fibers were isolated from BAMby centrifugation. BAM was dissolved in water at 2 mg/ml. The solutionwas then centrifuged at 180g for 10 min. The pellet, consisting of cellwall fibers, was harvested and washed three times with water beforebeing dried. Since BAM has gone through alcohol precipitation, thesefibers are therefore similar to those alcohol insoluble residues orsolids (AIS) that are commonly prepared for extraction of pectins fromother plant tissues.

The non-alcohol-treated fibers include the crude pulp and rind fibers.Crude pulp fibers were those retained by the coarse filtration duringproduction of BAM and other pulp-based products. They are the same asthose found in BAM, except for being larger in size and notalcohol-treated. They were collected with a no. 18 sieve (1 mm opening)with minimal loss and washed three times with water. The green rind,accounting for ˜60% wet weight of the whole leaf, are generallydiscarded as waste by manufacturers. It contained the green rind properas well as some pulp left behind after filleting. The fibers wereisolated from them in a similar way to those from pulp followinghomogenization. They were washed extensively, at least three times, withwater, then dried and stored at room temperature (“RT”) before beingused for pectin extraction.

Extraction

The chelating agent EDTA was used for extraction of Aloe pectins fromthe cell wall fibers. The fibers were suspended in water at 0.2-2%(w/v). The EDTA stock solution was prepared at 0.5 M and a pH of 7.0 or8.0 and added to the fiber suspension. The final concentration of EDTAused was 25 mM. The final pH of the fiber suspensions was adjusted withNaOH to the indicated values. The extraction was done with stirring ateither RT or with high temperature (“HT”) of about 80° C., or in asequential manner—RT extraction followed by HT extraction. HT wascarried out up to 80° C. and then stopped before the separation step. Inthe sequential extraction, the remaining fibers following the RTextraction were re-suspended to the same volume in water without washingand fresh EDTA was added at the same concentration as for the RTextraction. Following extraction, remaining fibers were removed bycentrifugation (500 g, 15 min) or by filtering with a no. 18 sievefollowed by gauze sponge filtering. The gauze sponges (4×4, 8 ply) wereused with three pieces together and set up in a disc filter frame. Thesponges were washed with water before use. The gauze sponge filtrationwas highly efficient in removing the residual small fibers after thesieve filtration. When necessary, the extract was passed through thesponge filter twice. The filtrate was essentially clear. Forquantitative studies on yields from sequential extraction, fibers werealways removed by centrifugation following the first round of extractionat RT. Alcohol (ethanol) was added to the clear supernatant or filtrateto a final concentration of 75% (v/v). The precipitates were collectedby centrifugation (500 g, 15 min) or with the no.18 sieve and washedtwice with 75% alcohol. The alcohol wash step was necessary to removeresidual EDTA. The precipitates were then pressed to remove alcohol,dried, and stored at RT before use.

The extraction of Aloe pectins with the chelating agent EDTA was foundto be highly efficient and a yield as high as 50% (w/w) could beobtained. The pectins obtained had an average galacturonic acid contentabove 70% (w/w). The pH was found to have a major effect on the pectinyield with EDTA extraction. A 5 mg/ml fiber suspension in water had a pHof ˜3.7 (3-4). The pH of the fiber suspension was 7.7 (7.5-8.0)following addition of pH 8.0 EDTA stock to a final concentration of 25mM. A pH of 6.4 (6.3-6.5) was obtained when a pH 7.0 EDTA stock solutionwas used to give a final concentration of 25 mM. The pH 5.0 was obtainedby using a pH 5.0 sodium acetate buffer at a final concentration of 20mM, a common condition for pectin extraction. It was found that therewas no major difference in yield following RT extraction at a pH from5.0 to 7.7. A major effect of pH, however, was found during HTextraction. A yield increase by >20% was noted at pH 7.7 as compared topH 5.0 or pH 6.4 during HT extraction of fresh fibers. Furthermore, anearly 2-fold increase in yield was noted when the remaining fibers fromthe first round of RT extraction were extracted under HT with fresh pH8.0 EDTA added as compared to using pH 7.0 EDTA. The pH values of thefiber suspensions did not change significantly at the end of RTextraction. However, after re-suspending in water and addition of freshEDTA, the pH (˜8.5) of the suspensions was actually higher than that ofEDTA stock solutions (pH 8.0). It was further found using the freshfibers under HT extraction that the pH 8.5 extraction did give a muchhigher yield, more than 2-fold higher than that at pH 5.0 and ˜40%higher than that at pH 7.7. Increasing the pH to 9.0, however, did notimprove the yield much further (<10%) as compared to pH 8.5. Ensuingexperiments also showed that a substantial increase (20%) in yield wasalso obtained with RT extraction at pH 8.5.

RT was less efficient than HT during extraction. The yield was similarbetween these two conditions provided the RT extraction was extended intime. The yield by RT extraction approached the maximum by ˜4 hrs.Further extension of the extraction time did not significantly improvethe yield. The yield of the second extraction with HT varied dependingon the length of the first RT extraction; therefore the yield with HTwould be higher if RT extraction was performed for only 1 hr, or lowerwhen the RT extraction was performed for 4 hrs or longer.

Repeated extraction under the same conditions produced a progressivelylower yield. The yield decreased by approximately half with eachextraction. The remaining fibers can therefore be suspended in half thevolume from the previous extraction.

EDTA and fiber concentrations also influenced the extraction efficiency.When 25 mM EDTA was used with a 2 mg/ml fiber suspension, a yieldbetween 50-60% could be obtained with a single extraction under HT. Whenusing a 5 mg/ml fiber suspension with the same EDTA concentration, theyield decreased to ˜30%. With the sequential room temperature to hightemperature extraction a combined yield of 40-50% could be readilyobtained. No difference in yield was noted between alcohol treated andnon-alcohol-treated fibers.

Other chelating agents were also considered for Aloe pectin extraction.Ammonium oxalate was not used because it is considered a toxic agent.Using sodium hexametaphosphate, a considerable yield was obtained;however, this agent was difficult to remove because of precipitateformation in alcohol solution and an acid (HCl or HNO₃) precipitationstep was required before the alcohol wash.

Other conditions were also examined for Aloe pectin extraction. Hotdilute acid and cold alkaline solutions are two other common conditionsfor pectin extraction. Both of them can cause extensive degradation.Commercial pectins from citrus and apple were extracted under the hotdilute acid condition. Using this condition for the Aloe pectin, the pHof fiber suspensions was adjusted to 1.5 with HCl followed by HT up to80° C. The yield obtained is much lower compared to using EDTAextraction. The extraction by HT in water alone yielded virtually noalcohol precipitable materials. Renault and Thibault (Renault andThibault, Carbohydrate Research, 1993, 244, pp. 99-114) reported thatextraction of apple and sugar beet fibers in PBS (pH 6.5) with HT (80°C.) generated a high yield similar to that by EDTA extraction. Usingthis condition, only a low yield was obtained from the Aloe Vera pulpfibers. Cold alkaline extraction was performed with 50 mM NaOH or 50 mMNa₂CO₃ at 4° C. The pH in suspension was 11.5 with 50 mM NaOH and 10.5with 50 mM Na₂CO₃. After 1 hr at 4° C., a very low yield was obtainedwith 50 mM Na₂CO₃. No alcohol precipitable materials were obtained with50 mM NaOH. When the extraction was done at RT for 1 hr, no yield wasobtained with either agent, suggesting that pectins are rapidly degradedunder these conditions.

Together, these results showed that extraction with EDTA at pH 7.0-8.5is the most efficient extraction method for Aloe pectin. With thesequential RT to HT extraction, a high yield (40-50%, w/w) could beobtained along with production of both HMW and LMW Aloe pectins. Theuniqueness of this extraction procedure was the higher pH (7.0-8.5)used. The reason behind this higher pH is that Aloe pectins arenaturally LM (see below) which are more resistant to β-elimination underalkaline conditions and EDTA functions most efficiently at a pH above7.0. In addition, EDTA is more soluble at a pH above 7.0 and cantherefore be more readily removed during alcohol precipitation and washsteps.

The green rind fibers produced a similar yield of pectin compared to thepulp fibers when extracted with the pH 8.0 EDTA. This rind pectin wasequally rich in Gal A. The amount of fibers obtained from the rind wasmore than 10 times higher than that from the pulp (per unit of leaves).This is consistent with the fact that the rinds consisted much smallercells as compared to the pulp. Together, these results indicated that avery large amount of Aloe pectin can be obtained from the rind portionof the leaf, which is currently discarded as waste materials by somemanufacturers.

To extract LM/HMW Aloe pectins with ETA at about room temperature, theworkable pH range appeared to be between about 5 and about 8.5,preferably about 8-8.5. To extract LM/LMW Aloe pectin with EDTA atelevated temperature (for example at about 80° C.), the workable pHranges appeared to be between about 5 and about 8.5, preferably about8.0. At pH of higher than 6.5, EDTA extraction of HM pectins from othersources at elevated temperature would lead to the degradation of theproducts. For the extraction of pectins from other plant sources usingEDTA or other chelating agents, the reported pH ranges are 4-6.5.

EXAMPLE 2 Pectin Purification by Ion Exchange Chromatography

The ion exchange chromatography was performed on a Pharmacia BiotechAKTA explorer chromatography system. The column was three PharnaciaHi-trap Q, 5 ml cartridges connected in series. Aloe pectins weredissolved in water at 1 mg/ml and loaded onto the column at a flow rateof 1 ml/min. After washing with 15 ml of water, bound materials wereeluted with a linear gradient of NaCl (0-1.0 M). The column eluant wasmonitored by UV absorbance at 215, 254, and 280 nm. Fractions containingpectin formed precipitates which were collected by low speedcentrifugation, pooled, and redissolved in water. They were thendesalted by passing through a Sephadex G-25 column. Thepectin-containing fractions were collected, dried, and stored at roomtemperature.

EXAMPLE 3 Calcium Gel Formation

Aloe pectins at various concentrations in water were mixed with calciumchloride solution at various concentrations along with commercial LM andHM pectins. After standing at RT for up to 24 hrs, the tubes wereinverted. If the sample flowed easily, it was considered that no gelformation occurred. If the sample did not flow or deform under its ownweight, gel formation had occurred. If the sample did not flow, butdeformed (i.e., the surface did not keep a straight line perpendicularto the side of the tube when tubes were held at a horizontal position),the system was considered as a soft gel. The results showed that Aloepectin obtained by either RT or HT extraction from either pulp or rindfibers formed firm gels in the presence of calcium as did the LM citruspectin and polygalacturonic acid. Under the same conditions, the HMcitrus pectin did not form gels. This is consistent with the fact thatthe Aloe pectin is a LM pectin. Pectins from citrus and apple arenaturally HM pectins. LM pectins are obtained by demethylating the HMpectins. Since no harsh conditions were applied during the extraction ofAloe pectins, especially with RT extraction, the Aloe pectin is anatural LM pectin.

With a 0.2% Aloe pectin solution, the minimum concentration of calciumchloride required for gel formation was determined to be 1-2 mM (50-100mg CaCl₂/g pectin). A gel could also be obtained using ZnCl₂ at asimilar concentration. With increasing concentrations of pectin and/orcalcium chloride, the gel became gradually firmer. It was noted that theHMW Aloe pectins formed gels more readily than LMW Aloe pectins in thatit took less time for gel formation and the gel seemed firmer.

Increasing the ionic strength facilitated the calcium gel formation. Thespeed of gel formation gradually increased with increasing NaClconcentrations (0-0.2M) after the addition of a fixed amount of calciumchloride.

EXAMPLE 4 Monovalent Cation-Based Gel Formation

Aloe pectins were dissolved in water at various concentrations. Thepectin solutions were mixed at RT with equal volumes of 0.3M NaCl(2×saline), 0.3M NaCl and 40 mM sodium acetate (pH 5.0), or 2×PBS (pH7.4). The final volumes were 1 or 2 ml. The tubes (12×75 mm) were thenplaced in a fridge at 4° C. or on ice (0° C.). The gel formation wasjudged as described in Example 3. The tubes were then returned to RT todetermine if the gel reverted back to solution. Various NaClconcentrations (0.05-1M) were tested for gel formation. The potassiumsalt (KCl) was also tested. The salt and pectin solutions were alwaysmixed in equal volumes (1:1). For determining the effect ofendo-polygalacturonase on the gel formation, pH 5.0 acetate buffer wasadded to pectin solutions to a final concentration of 20 mM. The enzymewas then added at indicated concentrations. After standing at RT for 30min, the solutions were mixed with equal volumes of 0.3M NaCl and thenplaced on ice. The gel formation was examined as above.

When an Aloe pectin solution in 0.15M NaCl (physiological saline) wascooled to 4° C., a gel was obtained. The gel was firm and free standingwhen kept at 4° C. just as the calcium gel; it turned quickly back tosolution when brought to RT (22° C.). This reversible solution-geltransition could be repeated many times by changing the temperature.

Unlike the gel formation in the presence of calcium which occurredefficiently with both HMW and LMW Aloe pectins, the monovalentcation-based gel formation only occurred efficiently with HMW Aloepectins obtained from either pulp or rind fibers. The sample AP 97-1 andsimilar ones, which had molecular weights of >1×10⁶ Da, could producefirm gels at concentrations as low as 1 mg/ml in the presence of 0.15MNaCl. Such gels were also clear when the pectin concentrations were 5mg/ml or less. With higher pectin concentrations (>5 mg/ml), gels werefirmer and slightly cloudy. With a 1 ml volume, a gel could form in ˜15min after the tube was placed on ice and returned to solution in aboutthe same time after it was brought back to RT. The gel, however, did notrevert back to solution at a temperature as high as 15° C. The gel couldform at pH 5.0 (in saline with 20 mM pH 5.0 sodium acetate) as well aspH 7.4 (in PBS).

The LMW (0.375-6.08×10⁵ Da) Aloe pectin only formed such gels at higherconcentrations (≧5 mg/ml). At 1 mg/ml, only soft gels could be obtainedwith some of the LMW samples in 0.15M NaCl. The smallest Aloe pectinsample (0.375×10⁵ Da) formed no gel at 1 mg/ml in 0.15M NaCl. A soft gelwas only obtained with this sample at a pectin concentration of 10 mg/mlin 0.2M NaCl. This suggests that the efficiency of the monovalentcation-based gel formation is dependent on the size of the pectinmolecules. Aloe pectin could be degraded by endo-polygalacturonase.Thus, 300 μl of 2 mg/ml AP97-1 pectin solution in 20 mM pH 5.0 sodiumacetate was digested with this enzyme at various concentrations beforemixing with an equal volume of 0.3M NaCl and placed on ice. The resultsshowed that the control (no enzyme added) formed a gel and the samplewith the highest enzyme concentration remained a solution. Between thecontrol and the highest enzyme concentration, the transition fromsolution to gel was evident, i.e., the gel became softer with anincrease in the enzyme concentration until a complete solution wasobtained at the highest enzyme concentration. This result indicates thatthe size of the Aloe pectin molecules is an important factor inmonovalent cation-based gel formation.

The gel formation was also dependent on the NaCl concentration. In 0.1MNaCl, only soft gels were obtained with samples like AP 97-1. The firmgels only formed in 0.15M and 0.2M NaCl. Whereas the gel formed at 0.15MNaCl was fully reversible when the gel was brought back to RT, the gelformed at 0.2M NaCl was not readily reversible, especially for the HMWAloe pectins. This gel formed with NaCl is referred to as pectin sodiumgel. After standing at RT for 1 hr or longer, syneresis often occurredwith the gel formed at 0.2M NaCl, i.e., the liquid was separated fromthe gel. With higher NaCl concentrations (≧0.4M), precipitates formed atRT. The precipitates were white and amorphous at high NaClconcentrations (0.6-1M) and appeared to be fine granules at 0.4M NaCl.

Such cold gelation is also sensitive to the species of monovalentcations used. With KCl (0.05-1M), no cold gel formation occurred,although precipitates were formed at higher KCl concentrations (≧0.4M)at RT.

Precipitation of pectins at high salt concentrations and RT has beenpreviously observed. However, such a reversible monovalent cation(NaCl)—based cold gelation under the physiological condition (0.15MNaCl, pH 7.4) has not previously been described with any other pectins.So far, no such gelling system has been identified with any otherpolymers or substances in literature. Using the commercialpolygalacturonic acid, LM and HM pectins, no such monovalent coldgelation was obtained.

EXAMPLE 5 Growth Factor Binding by Pectin

Generically, a heparin-binding growth factor (“HBGF”) is mixed with apectin in a solution of water, buffer, or saline to give the resultantpectin/heparin-binding growth factor (“PHBGF”). The mixture can bestored at a temperature range of between about −80° C. and about 37° C.,preferably between about −80° C. and about 22° C., and more preferablybetween about −80° C. and about 4° C. Alternatively, the mixture can belyophilized and stored at the same temperature ranges. The concentrationor amount of pectin in the mixture can range from about 0.001 mg/ml toabout 40 mg/ml, preferably from about 0.01 mg/ml to about 20 mg/ml, andmore preferable from bout 0.05 mg/ml to about 10 mg/ml. Theconcentration or amount of HBGH in the mixture can range from about 0.01ng/ml to about 500,000 ng/ml, and preferably from about 0.1 ng/ml toabout 250,000 ng/ml, and more preferably from about 1 ng/ml to about100,000 ng/ml.

EXAMPLE 6 Growth Factor Binding by Aloe Pectin

Pectin-rich cell wall fibers were isolated from BAM or from homogenizedfresh Aloe Vera inner gel. BAM was suspended in deionized water at 2mg/ml. The solutions were stirred at room temperature (“RT”) for 3 hrsor at 4° C. overnight. They were then centrifuged at low speed (1000 rpmor 180 g) for 10 min (Beckman TJ-6). The pellet or cell wall fiber wascollected, washed once with deionized water, and dried (Centrivap,Labconco). For isolation of cell wall fibers from fresh inner gel, AloeVera leaves were cleaned by washing with water and filleted to removethe outer green rind. The clear inner gel was homogenized and cell wallfibers were isolated by centrifugation at 500 g for 15 min. They werewashed once with 75% alcohol and then three times with deionized water.The isolated cell wall fibers were then further homogenized to ensurethey were small enough (˜200 μm) so that they could be handled in aconsistent manner through pipette tips. They were then dried before use.These fibers were rich in Aloe pectin (˜50%, w/w). They were used in thebinding assay and also for pectin extraction. Cell wall fibers were alsoisolated from the fresh rind in the same manner and used for pectinextraction.

Aloe pectin was extracted from the inner gel or rind cell wall fibers asdescribed in Example 1. Briefly, the extraction was performed with thechelating agent (EDTA) either at RT or with heating (80° C.) and at a pHof 7.5-8.5. Pectin was precipitated with ethanol (75%, finalconcentration) and washed twice with 75% ethanol. It was dried andstored at RT. Extraction at RT produced the high-molecular-weight (HMW;0.785-1.36×10⁶ Da) Aloe pectin and the heating extraction produced thelow-molecular-weight (LMW; 0.375-6.08×10⁵ Da) one.

For binding assays, proteins were dissolved in TN buffer (25 mM Tris,0.15M NaCl, pH 7.4) at 100 μg/ml. Proteins (1 μg) were mixed with 20 μgof the inner gel cell wall fibers in a 200 μl reaction volume. BSA (20μg), which was shown to be a non-binding protein, was also added to thereaction mixture to prevent non-specific binding. The mixtures weregently vortexed and incubated at 37° C. for 1 hr with gentle vortexingonce every 20 min. The cell wall fibers were then pelleted at 500 g for5 min and washed three times with TN buffer. They were then suspended ingel loading buffer and kept in a boiling water bath for 3 min beforeseparation by SDS-polyacrylamide gel electrophoresis. A 15% gel wasused. The protein bands were visualized by Coomassie blue staining. Fordensitometry analysis of the protein bands, the Coomassie blue-stainedgel images were acquired using GS-5000 digital imaging system (AlphaInfotech Co.) and the optical densities (OD) of protein bands weremeasured using the NIH Image program (version 1.6; National Institute ofHealth). A positive binding was identified by detection of the proteinband from the binding reaction with an OD value at least 2 times higherthan that of the control (i.e. protein alone).

For inhibition assays, various amounts of soluble Aloe pectin or heparinprepared in TN buffer were added to the binding reaction. The rest ofthe steps were carried out as described above.

The results showed that Aloe pectin bound to aFGF, bFGF, KGF, andTGF-β1, but not to IL-8, IL-1β, IFN-γ, EGF, TGF-α, TNF-α, PDGE, C3,fibronectin or BSA (Table 1). aFGF, bFGF, and KGF belong to FGF familyand TGF-β1 belongs to TGF-β family. Members of these two growth factorfamilies are all heparin-binding. aFGF, bFGF, and KGF and TGF-β1 aresome examples of the pectin/heparin-binding growth factors. Thus, theseresults showed that Aloe pectin bound to the heparin-binding growthfactors. The binding to the heparin-binding proteins was selective sinceAloe pectin did not bind to other heparin-binding proteins tested (IL-8,IFN-γ, and fibronectin). However, no binding was detected with any ofnon-heparin-binding proteins tested (IL-1β, EGF, TGF-α, TNF-α, PDGF, C3,or BSA). The binding to bFGF and KGF was particularly strong since ˜0.5μg out of the 1 μg protein used was bound under the experimentalconditions used. The results with bFGF are shown in FIG. 1. The bindingwas effectively inhibited by soluble Aloe pectin. Both LMW and HMW Aloepectins from either inner gel or outer rind inhibited the binding. Thisconfirmed that the binding to the pectin-rich cell wall fibers wasmediated by the pectin molecules (FIG. 1). The binding was alsoinhibited by heparin (FIG. 1), being consistent with the heparin-bindingnature of these growth factors.

EXAMPLE 7 Growth Factor Binding by Other Pectins

Various commercial pectins were used in the inhibition assay todetermine if they have similar binding activities. They includedde-esterified pectin (i.e. polygalacturonic acid) prepared from citruspectin, citrus LM pectin (DM, 28%), and citrus HM pectins with a DM of64% or 92%. All of them produced cloudy solutions when dissolved. Beforeuse, they were centrifuged at 25,000 g for 30 min to remove theinsoluble materials. The soluble pectins were precipitated with ethanol(75%, final concentration) and washed once with 75% ethanol. They werethen dried and stored at room temperature.

The inhibition assays were carried out with bFGF as described above.Aloe pectin exhibited an inhibition of 52% at 20 μg and 89% at 200 μg(FIG. 2). No inhibition (<20%) was observed with LM (DM, 28%) or HM (DM,64% or 92%) pectins at either 20 or 200 μg (FIG. 2), indicating thatthese pectins do not bind to the growth factor. A weak inhibition,however, was observed with the polygalacturonic acid as compared to Aloepectin, i.e., no inhibition (<20%) was observed at 20 μg, but asignificant inhibition (67%) was observed at 200 μg (FIG. 2). Thisindicates that polygalacturonic acid can also bind to the growth factoralthough not as strongly as the Aloe pectin.

EXAMPLE 8 Protection of Growth Factor against Protease Digestion

Heparin binding has been shown to stabilize growth factors againstinactivation by proteases. To determine if the pectin binding confersany protective effect on the growth factors, bFGF was subjected totrypsin digestion in the presence or absence of Aloe pectin or inner gelcell wall fibers. bFGF (0.5 μg) was mixed with Aloe pectin or inner gelcell wall fibers (0-16 μg) in a 15 μl reaction volume and incubated at37° C. for 10 min followed by the addition of trypsin (0.5 μg). Themixtures were incubated at 37° C. for 1 hr and then mixed with the gelloading buffer and subjected to electrophoresis as described above.

The results showed that under the conditions used, bFGF (0.5 μg) wasalmost completely digested by trypsin in the absence of Aloe pectin,i.e., only 3.5% of bFGF was still intact (FIG. 3). However, in thepresence of Aloe pectin, the bFGF was protected in a dose-dependentmanner. A 27, 32, 40, or 47% protection (remaining/starting×100%) wasobtained with 4, 8, 12, or 16 μg of Aloe pectin (FIG. 3). When BSA, anon-binding protein, was used instead of bFGF, no such protection effectwas observed. This indicates that binding by Aloe pectin does confer astabilizing effect on the growth factor. The same dose-dependentprotection of bFGF against trypsin digestion was also observed withinner gel cell wall fibers.

The protection by polygalacturonic acid was weaker as compared to Aloepectin, i.e., the amount of bFGF protected by polygalacturonic acid wasmuch less than that by Aloe pectin at any given amount used.

EXAMPLE 9 Growth Factor Binding by Pectin Calcium Gel Beads

In stabilizing a pectin/heparin-binding growth factor (“PHBGH”) with acalcium gel bead, the PHBGH is mixed with pectin calcium gel beadssuspended in a buffered or non-buffered solution. The mixture is shakenat between about 0° C. and about 37° C., preferably between about 0° C.and about 30° C., and more preferably between 4° C. and about 22° C.,for a period of from 5 minutes to about 24 hours, preferably from about15 minutes to about 6 hours, and more preferably between about 30minutes to about 2 hours. The treated beads are then isolated, andstored in a solution. Alternatively, the beads are dried bylyophilization. The concentration or amount of PHBGH that can be used inthe mixture can range from about 0.1 ng/ml to about 500,000 ng/ml,preferably from about 1 ng/ml to about 250,000 ng/ml, and morepreferably from about 10 ng/ml to about 100,000 ng/ml. The concentrationor amount of the pectin for the making of gel beads ranges from about 1mg/ml to about 40 mg/ml, preferably from about 5 mg/ml to about 30mg/ml, and more preferably from about 10 mg/ml to about 20 mg/ml. Thesize of the pectin calcium gel beads used in the mixture can vary fromabout 3-4 mm to about 10 μm, depending on the dripping method used. Theresultant treated beads can be stored in a temperature range of betweenabout −80° C. and about 37° C., preferably between about −20° C. andabout 22° C., and more preferably between about 0° C. and about 4° C.

Aloe pectin, like other LM pectins, is capable of forming a gel in thepresence of calcium (CaCl₂). The Aloe pectin calcium gel beads wereprepared by dripping the Aloe pectin solution (10 mg/ml) into a 200 mMCaCl₂ solution under stirring. Depending on the dripping mechanism, beadsize could vary from about 3-4 mm to <10 μm. Dripping through a syringeneedle gives larger beads, whereas spraying with a fine sprayer producessmaller beads. The beads formed in this experiment were ˜1 mm indiameter. These beads were then used in the binding assay as describedin Example 6 except that the binding reaction mixtures were kept at roomtemperature with constant shaking for 1 hr.

The results showed that these beads bound to bFGF (FIG. 4). The bindingwas inhibited by the soluble Aloe pectin. The same result was alsoobtained with KGF. This suggests that such beads can be loaded with thegrowth factor. Thus, pectin beads prepared with Aloe pectin can be usedas a delivery vehicle for the PHBGH.

EXAMPLE 10 Encapsulation of Growth Factors in Pectin Calcium Gel andMonovalent Cation-Based Gel

Generically, in stabilizing a pectin/heparin-binding growth factor(“PHBGF”) with a monovalent cation-based gel, such as an Aloe pectinsodium gel, the PHBGF is mixed with a pectin, such as an Aloe pectin, ina buffered or non-buffered physiological saline (about 0.15M NaCl). Themixture is kept at between about 0° C. and about 4° C. so that themonovalent cation-based gel can form. The concentration or amount ofPHBGF that can be used in the mixture is as described in Example 5. Theconcentration or amount of the pectin that can be used in the mixtureranges from about 0.1 mg/ml to about 20 mg/ml, preferably from about 0.5mg/ml to about 10 mg/ml, and more preferably from about 1 mg/ml to about5 mg/ml. The resultant mixture can be stored in a temperature range ofbetween about −80° C. and about 37° C., preferably between about −20° C.and about 22° C., and more preferably between about 0° C. and about 4°C.

Generally, in stabilizing a PHBGF with pectin calcium gel, the PHBGF ismixed with a pectin, such as an Aloe pectin, in a solution of calciumsalt of from about 0.1 mM to about 500 mM, preferably from about 0.5 mMto about 100 mM, and more preferably from about 1 mM to about 20 mM.Alternatively, the mixture of pectin and PHBGF was dripped into a 200 mMCaCl₂ bath to form beads as described in Example 9. The concentration oramount of PHBGF that can be used in the mixture is as described inExample 5. The concentration or amount of the pectin that can be used inthe mixture ranges from about 0.1 mg/ml to about 40 mg/ml, preferablyfrom about 0.5 mg/ml to about 30 mg/ml, and more preferably from about 1mg/ml to about 20 mg/ml. The resultant mixture can be stored in atemperature range of between about −80° C. and about 37° C., preferablybetween about −20° C. and about 22° C., and more preferably betweenabout 0° C. and about 4° C.

Aloe pectin solution in deionized water or physiological saline (0.15MNaCl) was mixed with bFGF so that the final pectin concentration was 10mg/ml and the final bFGF concentration was 100 μg/ml. The mixtures werethen dripped into a 200 mM CaCl₂ solution to make the calcium gel beads.

The beads were readily formed in the presence of bFGF. Using bovineserum albumin (“BSA”), beads could be obtained at a BSA concentration of10 mg/ml. The beads could also be obtained by dripping theBSA-containing pectin solution into a 200 mM ZnCl₂ solution. Such gelbead formation was also achieved with the de-esterified citrus pectin(polygalacturonic acid), although a higher concentration (20 mg/ml) ofpolygalacturonic acid had to be used. Growth factors/cytokines aregenerally used at a concentration well below 100 μg/ml. Thus, theprotein concentration is not a limiting factor for preparing suchcalcium gel beads. Compared to loading the pre-formed pectin beads withgrowth factors described in Example 9, entrapment in calcium gel likelyprovides a more protective delivery device since growth factors areentrapped inside the gel matrix. This form of delivery device is moresuitable for oral use.

For monovalent cation-based gel formation, the pectin and bFGF mixturewas prepared in physiological saline (0.15M NaCl) with a HMW Aloe pectinat a concentration of 1-2 mg/ml. The bFGF concentration was the same asabove (100 μg/ml). A gel formed after the mixture was kept at 0-4° C.for 10-30 min dependent on the volume of the mixture. Using BSA, such agel could be obtained at a BSA concentration of 20 mg/ml. Thus, thesepectin/heparin-binding growth factors can be stored in a gel. This notonly provides a direct stabilizing effect but also reduces the chancefor aggregate formation. The gel was reversible, changing quickly backto solution when brought back to room temperature (22° C.) and thusallowing the agent to be used in solution form.

EXAMPLE 11 Affinity Chromatography With Inner Gel Cell Wall Fiber As APectin-Containing Matrix

To assess the binding strength between Aloe pectin and the growthfactors, affinity chromatography was performed with inner gel cell wallfiber as a pectin-containing matrix. Inner gel cell wall fibers (0.4 ml)were packed into a 5 ml-column. The column was washed extensively withTN buffer. The bFGF (8 μg in 200 μl) was loaded onto the column, whichwas then washed with 8 ml TN buffer at a flow rate of 6 ml/hr. Theelution was performed stepwise with 0.4 ml each of 0.15, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, and 2.0M NaCl. The eluate was thenanalyzed by gel electrophoresis to detect bFGF as described above.

The results showed that bFGF was eluted at 0.5-0.7M NaCl with the peakat 0.6 M (FIG. 5). This showed that the binding strength of bFGF to Aloepectin was moderate and weaker than that to heparin; bFGF is eluted froma heparin column at 1.4-1.6M NaCl and aFGF at 0.8-1.0M NaCl (0.3-0.4MNaCl is considered weak binding and >1.0M NaCl is considered strongbinding; Conrade, (1998), Heparin-binding Proteins, Academic Press, SanDiego, pp. 197-199).

The weaker affinity to Aloe pectin as compared to heparin most likelymakes it more suitable for using Aloe pectin as a stabilizer anddelivery vehicle, i.e., once the growth factor bound to Aloe pectincontacts the glycosaminoglycan (heparin) on the cell surfaces, it willbind to the latter, its natural higher-affinity ligand for initiating astimulatory effect on cells.

EXAMPLE 12 Coupling of Pectin to a Support for Isolation ofPectin/Heparin-Binding Proteins

The carboxyl group of the Gal A residue in pectin is used for couplingof pectin to an amine-containing support with a carbodiimide compoundEDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide). Theamine-containing support can be the commercially availableaminohexylsepharose (Amersham Pharmacia Biotech) or diaminodipropylamineimmobilized on 3M Emphaze support (Pierce, Rockford, Ill.). Pectin (1-20mg/ml) is prepared in a buffer (0.1 M Mes, 0.15M NaCl, pH 4.7) and mixedwith the matrix. The EDC (0.1-20 mg/ml) is added to initiate thecoupling which lasts for 3 hrs at RT under shaking. After being washedwith 1M NaCl, the matrix is ready for use.

EXAMPLE 13 Pharmaceutical Formulation of Pectin/Heparin-Binding GrowthFactor

One embodiment of a pharmaceutical formulation of apectin/heparin-binding growth factor can be made by mixing and blendingthe following ingredients:

A pectic substance in a amount ranging from about 0.001 mg/ml to about40 mg/ml;

A pectin/heparin-binding growth factor (“PHBGF”) in an amount rangingfrom about 0.1 ng/ml to about 100,000 ng/ml. The PHBGF can be selectedfrom the group consisting of an acidic fibroblast growth factor, a basicfibroblast growth factor, a keratinocyte growth factor and atransforming growth factor β1;

A thickener in an amount ranging from about 20 mg/ml to about 150 mg/ml.The thickener can be selected from the group consisting of ahydroxyethyl cellulose, a Karaya gum, a cationic polyacrylamidecompound, and a sodium carboxyl methyl cellulose;

An optional preservative in an amount ranging from about 0.02 mg/ml toabout 5 mg/ml. The optional preservative can be selected from the groupconsisting of a paraben, a benzothonium halide, a benzoic acid, abenzoate, a sorbic acid, a sorbate, a sodium borate, and an antibiotic.

An optional dispersant in an amount ranging from about 10 mg/ml to about150 mg/ml. The optional dispersant can be selected from the groupconsisting of PVP, vinyl acetate copolymer, and polyvinylpyrrolidone.

The remaining is water, saline or a buffer solution.

Although the invention has been described with reference to thepresently-preferred embodiments, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

What is claimed is:
 1. A composition of a stabilized mammalianpectin/heparin-binding protein comprising a mammalianpectin/heparin-binding protein and a pectic substance having a degree ofmethylation of less than 50% in an amount effective to stabilize themammalian pectin/heparin-binding protein.
 2. The composition of claim 1,wherein the pectic substance is soluble in water.
 3. The composition ofclaim 1, wherein the pectic substance comprises Aloe Vera inner gel cellwall fiber.
 4. The composition of claim 1, wherein the pectic substancecomprises an Aloe pectin.
 5. The composition of claim 1, wherein thepectic substance is a pectin calcium gel, a pectin zinc gel, or amixture thereof.
 6. The composition of claim 1, wherein the mammalianpectic/heparin-binding protein comprises a mammalianpectin/heparin-binding growth factor.
 7. The composition of claim 1,wherein the pectin substance comprises a pectin-rich substance.
 8. Thecomposition of claim 1 further comprising a thickener.
 9. Thecomposition of claim 1, wherein the mammalian pectin/heparin-bindingprotein comprises a mammalian pectin/heparin-binding growth factor. 10.The composition of claim 9, wherein the mammalian pectic/heparin-bindinggrowth factor comprises an acidic fibroblast growth factor, a basicfibroblast growth factor, a keratinocyte growth factor, a transforminggrowth factor β1, or a mixture thereof.
 11. A composition of astabilized mammalian pectin/heparin-binding protein comprising amammalian pectin/heparin-binding protein, a pectic substance in anamount effective to stabilize the mammalian pectin/heparin-bindingprotein, and a solution of an inorganic salt.
 12. The composition ofclaim 11, wherein the inorganic salt is a calcium salt at aconcentration from about 0.1 mM to about 100 mM or a zinc salt at aconcentration from about 0.1 mM to about 100 mM, or a combinationthereof.
 13. The composition of claim 11, wherein the inorganic saltcomprises a sodium chloride maintained at a temperature from about 0° C.to about 4° C., at a concentration from about 0.05M to about 0.3M. 14.The composition of claim 11, wherein the mammalianpectin/heparin-binding protein comprises a mammalianpectin/heparin-binding growth factor.
 15. The composition of claim 14,wherein the pectin/heparin-binding growth factor is an acidic fibroblastgrowth factor, a basic fibroblast growth factor, a keratinocyte growthfactor, a transforming growth factor β1, or a mixture thereof.
 16. Thecomposition of claim 11, further comprising a thickener.
 17. Acomposition of a stabilized pectin/heparin-binding growth factorcomprising a pectin/heparin-binding growth factor and an Aloe pectin inan amount effective to stabilize the pectin/heparin-binding growthfactor.
 18. The composition of claim 17, wherein the Aloe pectin issoluble or insoluble in water.
 19. The composition of claim 17, whereinthe Aloe pectin comprises a pectin calcium gel.
 20. The composition ofclaim 17, wherein the Aloe pectin comprises a pectin sodium gel.
 21. Thecomposition of claim 17 further comprising a thickener.
 22. Apharmaceutical formulation comprising: a pectin/heparin-binding growthfactor; a pectic substance having a degree of methylation of about 64%or less in an amount effective to stabilize the pectin/heparin-bindinggrowth factor mixed with the pectin/heparin-binding growth factor; and athickener added to the mixture of the pectin/heparin-binding growthfactor and the pectic substance.
 23. The pharmaceutical formulation ofclaim 22, wherein the pectic substance is soluble or insoluble in water.24. The pharmaceutical formulation of claim 22, wherein the pecticsubstance comprises Aloe Vera inner gel cell wall fiber.
 25. Thepharmaceutical formulation of claim 22, wherein the pectic substancecomprises Aloe pectin.
 26. The pharmaceutical formulation of claim 22,wherein the pectic substances is a pectin calcium gel, a pectin zincgel, or a mixture thereof.
 27. The pharmaceutical formulation of claim22, wherein the pectic substance comprises pectin sodium gel.
 28. Thepharmaceutical formulation of claim 22, wherein thepectin/heparin-binding growth factor is an acidic fibroblast growthfactor, a basic fibroblast growth factor, a keratinocyte growth factor,a transforming growth factor β1, or a mixture thereof.
 29. Thepharmaceutical formulation of claim 22, wherein the pectic substance hasa concentration ranging from about 0.01 mg/ml to about 40 mg/ml.
 30. Thepharmaceutical formulation of claim 22, wherein thepectin/heparin-binding growth factor has a concentration ranging fromabout 10 ng/ml to about 100,000 ng/ml.
 31. The pharmaceuticalformulation of claim 22, wherein the thickener has a concentrationranging from about 20 mg/ml to about 150 mg/ml.
 32. A pharmaceuticalformulation comprising: a pectin/heparin-binding growth factor in anamount of from about 10 ng/ml to about 100,000 ng/ml, thepectin/heparin-binding growth factor is selected from the groupconsisting of an acidic fibroblast growth factor, a basic fibroblastgrowth factor, a keratinocyte growth factor, a transforming growthfactor β1, or a mixture thereof, a pectic substance having a degree ofmethylation of about 64% or less in an amount of from about 0.01 mg/mlto about 40 mg/ml mixed with the pectin/heparin-binding growth factor,wherein the pectic substance is selected from the group consisting ofAloe pectin, Aloe Vera inner gel cell wall fiber, Aloe pectin calciumgel, and Aloe pectin sodium gel; and a thickener in an amount of fromabout 20 mg/ml to about 150 mg/ml added to the mixture of thepectin/heparin-binding growth factor and the pectic substance, thethickener is selected from the group consisting of hydroxyethylcellulose, karaya gum, a cationic polyacrylamide compound, and sodiumcarboxyl-methylcellulose.
 33. A method of stabilizing apectin/heparin-binding protein, the method comprising adding a pecticsubstance having a degree of methylation of about 64% or less to apectin/heparin-binding protein in vitro, in vivo or ex vivo.
 34. Themethod of claim 33, wherein the pectic substance is an Aloe pectin, anAloe Vera inner gel cell wall fiber, an Aloe calcium pectate, an Aloepectin calcium gel, an Aloe sodium pectate, or an Aloe pectin sodiumgel.
 35. The method of claim 33, wherein the pectin/heparin-bindingprotein comprises pectin/heparin-binding growth factor.
 36. The methodof claim 35, wherein the pectin/heparin-binding growth factor is anacidic fibroblast growth factor, a basic fibroblast growth factor, akeratinocyte growth factor, a transforming growth factor β1, or amixture thereof.
 37. A method of stabilizing a pectin/heparin-bindingprotein, the method comprises: adding an Aloe pectin having a degree ofmethylation of about 64% or less to a pectin/heparin-binding protein inthe presence of a sodium chloride solution maintained at a temperaturefrom about 0° C. to about 4° C., the sodium chloride solution having aconcentration of from about 0.05M to about 0.3M.
 38. The method of claim37, wherein the pectin/heparin-binding protein is pectin/heparin-bindinggrowth factor.
 39. The method of claim 37, wherein thepectin/heparin-binding growth factor is an acidic fibroblast growthfactor, a basic fibroblast growth factor, a keratinocyte growth factor,a transforming growth factor β1, or a mixture thereof.
 40. The productprepared by the method of claim
 37. 41. The product prepared by themethod of claim
 38. 42. The product prepared by the method of claim 39.43. A method of stabilizing a pectin/heparin-binding protein, the methodcomprises: adding a pectic substance having a degree of methylation ofabout 64% or less to a pectin/heparin-binding protein in the presence ofa calcium salt solution, a zinc salt solution or a combination thereof,the calcium salt solution or the zinc salt solution having aconcentration of from about 0.1 mM to about 100 mM.
 44. The method ofclaim 43, wherein the pectin/heparin-binding protein comprisespectin/heparin-binding growth factor.
 45. The method of claim 44,wherein the pectin/heparin-binding growth factor is an acidic fibroblastgrowth factor, a basic fibroblast growth factor, a keratinocyte growthfactor, a transforming growth factor β1, or a mixture thereof.
 46. Theproduct prepared by the method of claim
 43. 47. The product prepared bythe method of claim
 44. 48. The product prepared by the method of claim45.
 49. A method of stabilizing a pectin/heparin-binding protein, themethod comprises: adding a pectin calcium gel wherein the pectin has adegree of methylation of about 64% or less to a pectin/heparin-bindingprotein, the pectin calcium gel having a pectin concentration of fromabout 0.01 mg/ml to about 40 mg/ml, and the pectin/heparin-bindingprotein having a concentration of from about 10 ng/ml to about 100,000ng/ml.
 50. The method of claim 49, wherein the pectin calcium gel is anAloe pectin calcium gel.
 51. The method of claim 49, wherein thepectin/heparin-binding protein is a pectin/heparin-binding growthfactor.
 52. The method of claim 51, wherein the pectin/heparin-bindinggrowth factor is an acidic fibroblast growth factor, a basic fibroblastgrowth factor, a keratinocyte growth factor, a transforming growthfactor β1, or a mixture thereof.
 53. The product prepared by the methodof claim
 49. 54. The product prepared by the method of claim
 50. 55. Theproduct prepared by the method of claim
 51. 56. The product prepared bythe method of claim
 52. 57. The composition of claim 1, wherein thepectic substance is insoluble in water.